Bacterial strain Rhodococcus aetherivorans VKM Ac-2610D producing nitrile hydratase, method of its cultivation and method for producing acrylamide

ABSTRACT

The invention relates to a bacterial strain belonging to the genus  Rhodococcus  which is a producer of a nitrile hydratase. The invention also relates to a method for producing acrylamide by hydration of acrylonitrile using a biomass of the bacterial strain and to a method of culturing the bacterial strain.

FIELD OF THE INVENTION

The group of inventions relates to a biotechnology and microbiologicalindustry and includes:

-   -   a new strain of bacterium Rhodococcus aetherivorans having a        high nitrile hydratase enzyme activity;    -   a method of its cultivation;    -   a method for producing concentrated solutions of acrylamide        using cells of the strain as a biocatalyst.

BACKGROUND OF THE INVENTION

The ability of microorganisms to transform carbonitriles tocorresponding amides was described in literature at the beginning of70^(th) years of the XX century. Nitrile hydratase enzyme catalyzingsuch reactions is inherent to a wide range of bacteria relating tovarious taxonomical groups. Representatives of the genus Rhodococcus areof practical interest with respect to the subject of the presentspecification. Large chemical and biotechnological companies of Japan,Korea, France, Russia, Germany, the USA and China use, inter alia, cellsof strains belonging to this genus as effective biocatalysts foracrylamide production.

Despite deep investigations of the process of the enzymatic hydrolysisof nitriles to corresponding amides and considerable successes in thefield of selection of strains producing nitrile hydratase, the industrydemand for new biocatalysts has not dropped. This is caused, on the onehand, by the efficacy and ecological safety of biotechnologicalproduction of amides, in particular acrylamide, and on the other hand bya high cost of earlier patented strains and technologies. Therefore inrecent years new microorganisms which were producers of a nitrilehydratase enzyme were isolated.

Known bacterial strains and methods for producing acrylamide using suchstrains, however, suffer from several drawbacks. Many strains are onlycapable of producing a maximum concentration of less than 40% ofacrylamide, and therefore the use of such strains is limited.

Another disadvantage of certain strains is the components that areexpensive and vary in composition, such as vitamins, peptone or yeastextract, must be included in their cultivation medium. Some strainsexhibit only low nitrile hydratase activity. To increase activity,additional steps such as removal of oxygen from the culture brothtogether with the enzyme activation during several days may be required.

Some strains require a cultivation medium that contains toxiccomponents. For instance, acetonitrile may be required in thecultivation medium, but acetonitrile is toxic, volatile, highlyinflammable and expensive.

SUMMARY

The disclosure relates, in part, to the bacterial strain Rhodococcusaetherivorans VKM Ac-2610D.

The disclosure relates, in part, to a method for producing acrylamide.

The disclosure relates, in part, to a method of culturing the bacterialstrain described herein.

The disclosure relates, in part, to a final product obtained by themethods described herein.

DETAILED DESCRIPTION

The present invention relates to a bacterial strain Rhodococcusaetherivorans VKM Ac-2610D.

In an embodiment, the bacterial strain Rhodococcus aetherivorans VKMAc-2610D is a producer of a nitrile hydratase.

The present patent application relates also to a method of cultivatingthe bacterial strain Rhodococcus aetherivorans VKM Ac-2610D.

The present application relates also to a method of producingacrylamide.

An object of the claimed group of inventions is the creation of a newbacterial strain having a high nitrile hydratase activity for use in theindustrial production of merchantable products.

An object of the claimed method of cultivating the strain is thedevelopment of a technology for the biomass production of the claimedstrain without a superfluous cost, moreover, if possible, with a costreduction compared with the existing similar methods that are used inindustry.

An object of the claimed method of producing the referred product by useof a new strain is the development of a technology for producingacrylamide at a predetermined concentration by use of the new strainwithout increasing the process cost and without additional complicationof process operations.

The present invention also relates to a method of culturing a bacterialstrain Rhodococcus aetherivorans VKM Ac-2610D, wherein cells of thestrain are cultured using a nutrient medium comprising

an aqueous phosphate buffer comprising sodium and potassium ions;

a source of carbon;

a source of nitrogen;

optionally an enzyme inducer;

optionally a cobalt salt;

a magnesium salt;

a zinc salt;

a calcium salt; and

a Fe(II) salt.

In an embodiment, the nutrient medium comprises

an aqueous phosphate buffer comprising sodium and potassium ions;

a source of carbon;

a source of nitrogen;

an enzyme inducer;

a cobalt salt;

a magnesium salt;

a zinc salt;

a calcium salt; and

a Fe(II) salt.

In an embodiment, the nutrient medium consists of

an aqueous phosphate buffer comprising sodium and potassium ions;

a source of carbon;

a source of nitrogen;

optionally an enzyme inducer;

optionally a cobalt salt;

a magnesium salt;

a zinc salt;

a calcium salt; and

a Fe(II) salt.

In an embodiment, the nutrient medium consists of

an aqueous phosphate buffer comprising sodium and potassium ions;

a source of carbon;

a source of nitrogen;

an enzyme inducer;

a cobalt salt;

a magnesium salt;

a zinc salt;

a calcium salt; and

a Fe(II) salt.

In an embodiment, the cells of the strain are cultured in the nutrientmedium.

In an embodiment, the cells of the strain are suspended in the nutrientmedium during the culturing.

In an embodiment, the pH of the nutrient medium is 6.3-8.3. In anembodiment, the pH of the nutrient medium is 7.0-7.4.

In an embodiment, the process is carried out at a temperature of 26-31°C. In an embodiment, the process is carried out at a temperature of28-30° C. In this context, the term “the process” should be understoodas referring to the process of culturing the cells of the strain usingthe nutrient medium.

In an embodiment, the cells of the bacterial strain are cultured withstirring.

In an embodiment, the cells of the bacterial strain are cultured withaeration.

In an embodiment, the cells of the bacterial strain are cultured untilthe growth of the cells of the bacterial strain enters the stationaryphase.

In an embodiment, the cells of the bacterial strain are cultured until apredetermined yield of cells of the bacterial strain is achieved.

The predetermined yield may vary e.g. depending on the amount of biomassthat should be obtained for the process of producing acrylamide, or onthe nitrile hydratase activity of the cells of the bacterial strain.

The achievement of a predetermined yield of cells may be determined byknown methods such as measurement of optical density of the culture ormeasurement of the dry weight of the cell mass. Optical density, orabsorbance, may be measured e.g. in a photoelectric colorimeter asdescribed below. Likewise, said methods may be used to determine whenthe growth of the cells of the bacterial strain enters the stationaryphase.

In an embodiment, the cells of the bacterial strain are cultured untilthe achievement of an optical density of the suspension of 36-40. Inthis context, the “suspension” should be understood as referring to thenutrient medium comprising the cells of the bacterial strain suspendedtherein.

The optical density of the suspension of 36-40 indicates that bacterialgrowth has reached the stationary phase. Herein and in the Examples, theoptical density is measured in a photoelectric colorimeter with athickness of optical layer of 5 mm at the wavelength 540 nm.Photoelectric colorimeters or spectrophotometers suitable for measuringoptical density are well known in the art.

In an embodiment, the process is carried out until the achievement of anoptical density of the suspension of 36-40.

In an embodiment, the process is carried out until the achievement of anoptical density of the suspension of 36-40 at a wavelength of 540 nm anda thickness of an optical layer 5 mm.

The aqueous phosphate buffer may be any agueous phosphate buffer knownin the art, provided it comprises sodium (Na⁺) and potassium (K⁺) ions.Said aqueous phosphate buffer may be prepared using various phosphatesalts of sodium and potassium. In an embodiment, the aqueous phosphatebuffer comprises a phosphate salt. In an embodiment, the aqueousphosphate buffer comprises a phosphate salt selected from the groupconsisting of Na₂HPO₄ NaH₂PO₄, KH₂PO₄, K₂HPO₄ and any mixtures thereof.In an embodiment, the aqueous phosphate buffer comprises Na₂HPO₄.12H₂Oand KH₂PO₄. The pH of the aqueous phosphate buffer may be selected so asto adjust the pH of the nutrient medium to 6.3-8.3 or to 7.0-7.4.

In an embodiment, the source of carbon is selected from the groupconsisting of glucose, cellobiose, fructose, galactose, maltose,mannose, sucrose, trehalose, ribose, glycerol, mannitol, sorbitol,salicin, inulin, citrate, pyruvate, succinate, fumarate and any mixturesthereof.

In an embodiment, the source of carbon is glucose.

In an embodiment, the nutrient medium comprises 20.0-60.0 g/l of thesource of carbon.

The source of nitrogen may be any source of nitrogen the cells of thebacterial strain are able to utilize. Many suitable sources of nitrogenare also capable of inducing nitrile hydratase activity. A skilledperson will therefore understand that a single compound may be includedin the nutrient medium both as a source of nitrogen and as an enzymeinducer. In an embodiment, the source of nitrogen is selected from thegroup consisting of carbamide, leucine, acetamide or any mixturethereof. In an embodiment, the source of nitrogen is carbamide.

In this context, the words “enzyme inducer” and “inducer” may be usedinterchangeably and refer to an agent capable of inducing nitrilehydratase activity. Several enzyme inducers capable of inducing nitrilehydratase activity are known in the art. In an embodiment, the enzymeinducer is carbamide. In an embodiment, the enzyme inducer is analiphatic amide. In an embodiment, the enzyme inducer is selected fromthe group consisting of propionamide, isobutyramide, acetamide and amixture thereof.

In an embodiment, the source of nitrogen and the inducer is carbamide.Carbamide (urea) may be utilized as a microorganism-friendly source ofnitrogen and is readily available for industrial purposes. It can alsofunction as an enzyme inducer.

In an embodiment, the nutrient medium comprises 10.0-24.0 g/l of thesource of nitrogen.

The cobalt salt may be any soluble cobalt salt. Cobalt ions are utilizedas cofactors for the nitrile hydratase enzyme. In an embodiment, thecobalt salt is CoCl₂, CoSO₄ or a mixture thereof. In an embodiment, thecobalt salt is CoCl₂.6H₂O, CoSO₄.7H₂O or a mixture thereof. In anembodiment, the nutrient medium comprises 0.04-0.085 g/l of the cobaltsalt.

The magnesium salt may be any soluble magnesium salt. Magnesium ionsprovided by the magnesium salt are utilized in transport functions ofthe cells of the bacterial strain. In an embodiment, the magnesium saltis MgSO₄. In an embodiment, the magnesium salt is MgSO₄.7H₂O. In anembodiment, the nutrient medium comprises 10.0-24.0 g/l of the magnesiumsalt.

The zinc salt may be any soluble zinc salt. Zinc ions provided by thezinc salt may be utilized in decomposition of the nitrogen source suchas carbamide. In an embodiment, the zinc salt is ZnSO₄. In anembodiment, the zinc salt is ZnSO₄.7H₂O. In an embodiment, the nutrientmedium comprises 0.08-0.4 g/l of the zinc salt.

The calcium salt may be any soluble calcium salt. Calcium ions providedby the calcium salt may be involved in the utilization of the carbonsource such as glucose. In an embodiment, the calcium salt is selectedfrom the group consisting of CaCl₂, CaHPO₄.2H₂O, Ca(H₂PO₄)₂.2H₂O,Ca₃(PO₄)₂, CaCl₂.2H₂O, CaCO₃, CaSO₄, Ca(C₃H₅O₃)₂.3H₂O,Ca(HOCH₂(CHOH)₄COO)₂.H₂O and any mixtures thereof. In an embodiment, thenutrient medium comprises 0.2-0.6 g/l of the calcium salt.

The Fe(II) salt may be any soluble Fe(II) salt. Ferrous ions provided bythe salt are utilized in respiration. In an embodiment, the Fe(II) saltis in a chelate complex form. In an embodiment, the Fe(II) salt is FeSO₄in a chelate complex form. In an embodiment, the Fe(II) salt isFeSO₄.7H₂O in a chelate complex form. In an embodiment, the nutrientmedium comprises 0.025-0.05 g/l of the Fe(II) salt.

In an embodiment, the magnesium salt is MgSO₄; the zinc salt is ZnSO₄;the calcium salt is selected from the group consisting of CaCl₂,CaHPO₄.2H₂O, Ca(H₂PO₄)₂.2H₂O, Ca₃(PO₄)₂, CaCl₂.2H₂O, CaCO₃, CaSO₄,Ca(C₃H₅O₃)₂.3H₂O, Ca(HOCH₂(CHOH)₄COO)₂.H₂O any any mixtures thereof; andthe Fe(II) salt is FeSO₄.7H₂O in a chelate complex form.

The source of carbon, the source of nitrogen, the inducer, and thecobalt salt may be introduced in the nutrient medium in one portion orin several portions during the culturing and cell growth.

The nutrient medium is simple and cheap to manufacture. It also allowsfor a high yield of biomass of the bacterial strain and high activity ofnitrile hydratase in the biomass of the bacterial strain.

In an embodiment, the nutrient medium does not comprise vitamins, aminoacids, peptones, plant extracts, yeast extracts and/or acetonitrile.

The produced biomass may subsequently be separated. Methods forseparating the produced biomass are well known in the art.

In an embodiment, the cells of the strain are seeded on a solid nutrientmedium and cultivated for a time period, then the biomass is washed out,and the resulting suspension is used for inoculation of a first vesselcomprising the nutrient medium, and the process is carried out during atime period with stirring to achieve an optical density of thesuspension in the range of 2-16 units at a wavelength of 540 nm and athickness of an optical layer 5 mm; then the resulting suspension isused for inoculation of a second vessel having a volume which is 10-100times larger than a volume of the first vessel, the second vesselcomprising new nutrient medium, to achieve optical density of 0.1-0.3 inthe second vessel; the process is continued for a time period withaeration until the achievement of an optical density of the suspensionof 36-40 and a pH of 7.5-7.8; then the produced biomass is separated.

In an embodiment, the cells of the strain are seeded on a solid nutrientmedium and cultivated for 24-48 hours, then the biomass is washed out,and a resulting suspension is used for inoculation of a first vesselcomprising the nutrient medium, and the process is carried out during24-48 hours with stirring to achieve an optical density of thesuspension in the range of 2-16 units at a wavelength of 540 nm and athickness of an optical layer 5 mm, then the resulting suspension isused for inoculation of a second vessel having a volume which is 10-100times larger than a volume of the first vessel, the second vesselcomprising new nutrient medium, to achieve optical density of 0.1-0.3 inthe second vessel; the process is continued for 48-120 hours withaeration until the achievement of an optical density of the suspensionof 36-40 and a pH of 7.5-7.8; then the produced biomass is separated.

The cells of the bacterial strain may be cultured as a one-stepcultivation, wherein the cells of the bacterial strain are cultured inthe nutrient medium until a first predetermined yield of cells isachieved. The cells of the bacterial strain may also be cultured as atwo-step cultivation, wherein the cells of the bacterial strain arecultivated in the nutrient medium in a first vessel and subsequentlyusing the resulting culture or suspension to inoculate a new nutrientmedium in a second vessel; the cells of the bacterial strain arecultured in the new nutrient medium until a second predetermined yieldof cells is achieved. The first and second predetermined yield need notbe the same. Typically, the first predetermined yield need not be ashigh as the second predetermined yield.

The two-step cultivation, wherein the cells of the strain are culturedin the first vessel in a first step and in the second vessel as a secondstep may allow for better adaptation of the bacterial strain to themedium and improved enzyme synthesis.

The solid nutrient medium may be e.g. a meat and peptone agar, LB mediumagar, a synthetic medium or any other solid nutrient medium which iscapable of supporting the growth of the bacterial strain Rhodococcusaetherivorans VKM Ac-2610D.

The biomass may be washed out e.g. with a sterile physiological solutionsuch as phosphate-buffered saline. The sterile physiological solutionmay have a pH of 7.0-7.4.

The stirring of the first vessel may be e.g. circular stirring at a rateof 140-160 rpm.

In this context, the term “new nutrient medium” should be understood asreferring to a fresh volume of the nutrient medium defined above.

The invention also relates to a method of cultivating the bacterialstrain Rhodococcus aetherivorans VKM Ac-2610D wherein cells of thestrain are seeded on a meat and peptone agar slope and cultivated for24-48 hours, then a biomass is washed out with a sterile physiologicalsolution having a pH of 7.0-7.4, and a resulting suspension is used forinoculating a first vessel comprising a nutrient medium of the followingcomposition, g/l:

Na₂HPO₄•12H₂O  6.06 KH₂PO₄ 1.3 glucose 10.0-20.0 carbamide 2.0-6.0MgSO₄•7H₂O 1.0 ZnSO₄•7H₂O 0.08-0.2  CaCl₂•2H₂O 0.2 CoCl₂•6H₂O 0.01-0.02FeSO₄•7H₂O in a chelate complex form  0.01-0.025 Distilled water to 1 lpH 7.0-7.4;

and the process is carried out during 24-48 hours at a temperature of28-30° C. with a circular stirring at a rate of 140-160 rpm to achieveoptical density of the suspension in the range of 2-16 units at awavelength 540 nm with a thickness of optical layer 5 mm. Then theresulting suspension is used for inoculating a second vessel having avolume which is 10-100 times larger than a volume of the first vessel,the second vessel comprising a new nutrient medium of the followingcomposition, g/l:

Na₂HPO₄•12H₂O 6.20 KH₂PO₄ 1.65 glucose 20.0-60.0 carbamide 10.0-24.0MgSO₄•7H₂O 0.8-1.0 ZnSO₄•7H₂O 0.08-0.4  a calcium salt 0.2-0.6 a cobaltsalt  0.04-0.085 FeSO₄•7H₂O in a chelate complex form 0.025-0.05 distilled water to 1 l pH 7.0-7.4;

to achieve optical density of 0.1-0.3 in the second vessel; the processis continued for 48-120 hours at a temperature of 26-31° C., an aerationof 0.5-1.0 volume of air/volume of medium per a minute until achievementof a suspension optical density of 36-40 and a pH of 7.5-7.8; then theobtained biomass is separated.

In an embodiment of the above described method of cultivating thebacteria strain Rhodococcus aetherivorans VKM Ac-2610D, the nutrientmedium in the second vessel may comprise one of the following salts as acalcium salt:

CaHPO₄.2H₂O

Ca(H₂PO₄)₂.2H₂O

Ca₃(PO₄)₂

CaCl₂.2H₂O

CaCO₃

CaSO₄

Ca(C₃H₅O₃)₂.3H₂O

Ca(HOCH₂(CHOH)₄COO)₂.H₂O;

and one of following salts as a cobalt salt: CoCl₂.6H₂O,

CoSO₄.7H₂O.

The present invention also relates to a method for producing acrylamideby hydration of ac-rylonitrile using a biomass of a strain belonging tothe genus Rhodococcus and having a nitrile hydratase activity, whereinthe hydration is carried out at a working concentration of acrylonitrilewhich is no more than 0.5%, using a biomass of the bacterial strainRhodococcus aetherivorans VKM Ac-2610D.

A final product containing acrylamide is obtained by the method.

In an embodiment, the hydration is followed by isolation of the finalacrylamide product.

The term “final acrylamide product” should be understood as referring toacrylamide contained in the final product. Methods that are suitable forisolating acrylamide from the final product are known in the art.

Acrylonitrile is a toxic compound, and a suitable working concentrationthereof that is not overly toxic to the bacterial strain should bemain-tained. A working concentration of acrylonitrile which is no morethan 0.5% is tolerated by the bacterial strain. Said workingconcentration may also allow for obtaining acrylamide in the finalproduct at a concentration of 45-49%. The working concentration may bemaintained by loading acrylonitrile in the reaction solution during thehydration reaction. A skilled person may select the workingconcentration of acrylonitrile e.g. so that the hydration rate or theamount of the final acrylamide product is optimal. In an embodiment, theworking concentration of acrylonitrile is in the range of 0.01-0.5%, or0.05-0.5%, or 0.1-0.5%.

In an embodiment, the working concentration of acrylonitrile is no morethan 0.5%, or in the range of 0.01-0.5%, or 0.05-0.5%, or 0.1-0.5% byweight based on the total weight of the reaction solution (w/w).

In this context, the term “working concentration” should be understoodas referring to the concentration in the reaction solution comprisingthe biomass of the bacterial strain and acrylonitrile. The reactionsolution may also comprise other components, such as water, acrylamideand/or any additives.

The term “reaction solution” should be understood as referring to thereaction mixture comprising the biomass of the bacterial strain andacrylonitrile. The reaction mixture may also comprise other components,such as water, acrylamide and/or any additives.

The amount of the biomass of the bacterial strain used may be selectede.g. so that the hydration rate or the amount of the final acrylamideproduct is optimal. The amount of the biomass of the bacterial strainmay depend e.g. on the nitrile hydratase activity of the cells of thebacterial strain.

In an embodiment, the biomass of the bacterial strain Rhodococcusaetherivorans VKM Ac-2610D is at about 100-1000 g, or 200-1000 g, or200-800 g, or 300-600 g, or less than 1000 g, or less than 500 g, orless than 400 g on a dry weight of strain per 1 ton of the finalproduct.

In an embodiment, the biomass of the bacterial strain Rhodococcusaetherivorans VKM Ac-2610D is at about 400-500 g on a dry weight ofstrain per 1 ton of the final product.

In this context, the term “the final product” should be understood asreferring to the reaction solution comprising the biomass of the strainand acrylamide. The final product may also comprise other components,e.g. water, minor amounts of acrylonitrile and/or any additives. Askilled person will understand that the acrylonitrile included in thereaction solution is hydrated into acrylamide by the biomass of thebacterial strain. In other words, the final product refers to the entirereaction solution in which the acrylonitrile added in the reactionsolution has been hydrated into acrylamide.

The term “biomass” should be understood as referring to a mass of cellsof the bacterial strain.

In an embodiment, the final product contains acrylamide at aconcentration of at least 40%; or at least 41%; or at least 42%; or atleast 43%; or at least 44%; or 40-55%; or 41-55%; 42-55%; or 43-55%; or44-55%.

In an embodiment, the final product contains acrylamide at aconcentration of at least 45%; or at least 46%; or at least 47%; or45-55%; or 45-54%; or 45-53%; or 45-52%; or 45-51%; or 45-50%; or45-49%; or 46-55%; or 46-54%; or 46-53%; or 46-52%; or 46-51%; or46-50%; or 46-49%; or 47-55%; or 47-54%; or 47-53%; or 47-52%; or47-51%; or 47-50%; or 47-49%.

In an embodiment, the final product contains acrylamide at aconcentration of at least 40%; or at least 41%; or at least 42%; or atleast 43%; or at least 44%; or 40-55%; or 41-55%; 42-55%; or 43-55%; or44-55%; or at least 45%; or at least 46%; or at least 47%; or 45-55%; or45-54%; or 45-53%; or 45-52%; or 45-51%; or 45-50%; or 45-49%; or46-55%; or 46-54%; or 46-53%; or 46-52%; or 46-51%; or 46-50%; or46-49%; or 47-55%; or 47-54%; or 47-53%; or 47-52%; or 47-51%; or47-50%; or 47-49% by weight on the basis of the total weight of thefinal product.

In an example corresponding to Example 6, in order to obtain acrylamideat a concentration of 47% in the final product, 757.2 g of acrylonitrilemay be introduced into a reactor, resulting in a total reaction mass of2157.2 g. Since all acrylonitrile is transformed by the cells of thebacterial strain to acrylamide, a final concentration of 47% ofacrylamide is obtained in the final product. The reaction would, in thisexample, include 1 g of biomass (dry weight).

In an embodiment, the hydration of acrylonitrile is carried out at atemperature of 10-23° C.

In an embodiment, the hydration of acrylonitrile is carried out at a pHof 6.8-8.4.

In an embodiment, the hydration of acrylonitrile is carried out at atemperature of 10-23° C. and at a pH of 6.8-8.4.

In an embodiment, a biomass of the bacterial strain is suspended inaqueous solution; and

acrylonitrile is added in the aqueous solution to form a reactionsolution.

In an embodiment, acrylonitrile is mixed in an aqueous suspension of abiomass of the bacterial strain Rhodococcus aetherivorans VKM Ac-2610Dto form a reaction solution; and hydration is carried out so that theworking concentration of acrylonitrile in the reaction solution ismaintained at no more than 0.5%.

In an embodiment, the method comprises the following steps:

a) a biomass of the bacterial strain Rhodococcus aetherivorans VKMAc-2610D is suspended in aqueous solution to obtain a suspension;

b) acrylonitrile is mixed in the suspension obtainable from step a) toform a reaction solution; and

c) hydration is carried out so that the working concentration ofacrylonitrile in the reaction solution is maintained at no more than0.5%.

In an embodiment, the working concentration of acrylonitrile in thereaction solution is main-tained at no more than 0.5% until a finalproduct containing acrylamide at a concentration of at least 45% isobtained.

In an embodiment of the method including the hydration of acrylonitrileusing a biomass of a strain belonging to the genus Rhodococcus, havingnitrile hydratase activity, and then isolation of the final product,i.e. acrylamide, the hydration is carried out at a working concentrationof acrylonitrile no more than 0.5%, using a biomass of the bacterialstrain Rhodococcus aetherivorans VKM Ac-2610D in an amount of about400-500 g on the dry weight basis of the strain per 1 ton of the finalproduct that is acrylamide at a concentration of 45-49%.

The present invention also relates to the final product obtainable byone or more embodiments of the method for producing acrylamide.

In an embodiment, the final product comprises a biomass of the bacterialstrain and acrylamide. The final product may further comprise othercomponents, e.g. water, minor amounts of acrylonitrile and/or anyadditives.

In an embodiment, the final product contains acrylamide at aconcentration of at least 40%; or at least 41%; or at least 42%; or atleast 43%; or at least 44%; or 40-55%; or 41-55%; 42-55%; or 43-55%; or44-55%.

In an embodiment, the final product contains acrylamide at aconcentration of at least 45%; or at least 46%; or at least 47%; or45-55%; or 45-54%; or 45-53%; or 45-52%; or 45-51%; or 45-50%; or45-49%; or 46-55%; or 46-54%; or 46-53%; or 46-52%; or 46-51%; or46-50%; or 46-49%; or 47-55%; or 47-54%; or 47-53%; or 47-52%; or47-51%; or 47-50%; or 47-49%.

In an embodiment, the final product contains acrylamide at aconcentration of at least 40%; or at least 41%; or at least 42%; or atleast 43%; or at least 44%; or 40-55%; or 41-55%; 42-55%; or 43-55%; or44-55%; or at least 45%; or at least 46%; or at least 47%; or 45-55%; or45-54%; or 45-53%; or 45-52%; or 45-51%; or 45-50%; or 45-49%; or46-55%; or 46-54%; or 46-53%; or 46-52%; or 46-51%; or 46-50%; or46-49%; or 47-55%; or 47-54%; or 47-53%; or 47-52%; or 47-51%; or47-50%; or 47-49% by weight on the basis of the total weight of thefinal product.

The present invention also relates to acrylamide obtainable from thefinal product.

The technical result provided by the claimed group of inventions.

The strain of bacteria Rhodococcus aetherivorans VKM Ac-2610D ischaracterized in that it grows on a simple organo-mineral medium and hasa high nitrile hydratase activity up to 332 U/mg at 20° C. or 521 U/mgat 25° C. Nitrile hydratase of the bacterial strain Rhodococcusaetherivorans VKM Ac-2610D is thermostable.

The strain was isolated from wastewaters of the acrylamide andpolyacrylamide manufacture and was not genetically modified. It isespecially important for its use as an industrial biocatalyst because alegislation of many countries limits the use of genetically modifiedmicroorganisms.

The developed method of cultivating a bacterial strain Rhodococcusaetherivorans VKM Ac-2610D makes it possible to have a high yield ofcells having high activity on a simple organo-mineral nutrient medium,which does not comprise expensive inducers, vitamins, amino acids andplant extracts. Since all the components of the nutrient medium arecommercially available, the cost of the industrial biocatalyst preparedon the basis of the claimed strain is reduced. The method forcultivation according to one or more embodiments is optimal for rapidgrowth of the bacterial strain. Furthermore, the method for cultivationaccording to one or more embodiments may be used to provide a biomass ofthe bacterial strain that can be used in the method for producingacrylamide according to one or more embodiments.

The method for producing acrylamide by its synthesis using the bacterialstrain Rhodococcus aetherivorans VKM Ac-2610D described in thisapplication is economically advantageous. Conditions of synthesis ofconcentrated solutions of acrylamide are optimized.

The claimed strain is isolated from wastewaters of the acrylamide andpolyacrylamide manufacture by a method of direct seeding on a selectivemedium.

The claimed strain of bacteria Rhodococcus aetherivorans is depositedunder the Budapest Treaty by Individual entrepreneur Sergey Kozulin inthe AllRussian Collection of Microorganisms (Russian Collection ofMicroorganisms (VKM), G.K. Skryabin Institute of Biochemistry andPhysiology of Microorganisms, Russian Academy of Sciences, ProspektNauki No. 5, Pushchino 142290 (Moscow Region), Russian Federation) withan accession number VKM Ac-2610D (identification reference given by thedepositor: Rhodococcus aetherivorans KTN26-1). The strain was receivedby the Depositary Authority on 20, Jul. 2012, and the deposit wasconverted into a deposit under the Budapest Treaty on 15, Nov. 2013. Thestrain is characterized by the following morphological, culture andbiochemical properties.

Morphological properties: a gram-positive strain having a life cycle ofRhodococcus, non-motile, non-sporeforming, is not encysting, isacidintolerant, aerobic.

Culture properties: it forms round smooth colonies on meat and peptoneagar, having from yellow-orange to red-orange color and a diameter of1-2 mm (after 72-96 hours). When grown on special media it dissociatesto R- , S- and M-forms.

Physiological properties: The strain is oxidase-negative, catalase- andphosphatase-positive, reduces nitrates into nitrites. It hydrolyzesstarch, Tween 60 and Tween 80; it does not hydrolyze cellulose, esculinand DNA. It grows at a pH of 5.5-9.5, optimal pH value being 7.2±0.2; ata temperature of 5-45° C., optimal value being 29±1° C. As a sole sourceof carbon it uses: a cellobiose, fructose, galactose, glucose, maltose,mannose, sucrose, trehalose, ribose, glycerol, mannitol, sorbitol, butit does not use dulcitol and inositol. The strain grows on a salicin,inulin, citrate, pyruvate, succinate, fumarate, but does not grow on agluconate. It utilizes meta- and para-hydroxybenzoic acids, isobutanol,2,3-butyleneglycol, monoethanol amine. It uses leucine and acetamide asa sole source of nitrogen.

Pathogenicity: the strain is not pathogenic.

On the basis of the above properties, according to Bergey's Manual ofDeterminative Bacteriology (Opredelitel bakteriy Berdgy, Moscow: Mir,1997) and restriction fragment length polymorphism analysis of a gene16S of a ribosomal ribonucleic acid, the strain is attributed to thegenus Rhodococcus, aetherivorans species.

The method of cultivating the bacterial strain Rhodococcus aetherivoransVKM Ac-2610D may be carried out as follows.

Cells of the strain Rhodococcus aetherivorans stored at temperature of4° C. in stabs of 0.4% agarized LB medium or meat and peptone broth maybe seeded on a meat peptone agar slope and cultivated for 24-48 hours.The obtained biomass may be washed out with a sterile physiologicalsolution having a pH of 7.0-7.4. The obtained suspension can be used forinoculation of a first vessel comprising a nutrient medium of thefollowing composition, g/l:

Na₂HPO₄•12H₂O  6.06 KH₂PO₄ 1.3 glucose 10.0-20.0 carbamide 2.0-6.0MgSO₄•7H₂O 1.0 ZnSO₄•7H₂O 0.08-0.2  CaCl₂•2H₂O 0.2 CoCl₂•6H₂O 0.01-0.02FeSO₄•7H₂O in a chelate complex form  0.01-0.025 Distilled water to 1 lpH 7.0-7.4;

The process can be carried out during 24-48 hours at a temperature28-30° C. with a circular stirring at a rate of 140-160 rpm to achieveoptical density of the suspension in the range of 2-16 units measured ina photoelectric colorimeter with a thickness of optical layer of 5 mm atthe wavelength 540 nm.

Then the resulting suspension may be used for inoculating a second(larger) vessel having a volume which is 10-100 times larger than avolume of the first vessel, e.g. a large flask or fermenter comprising anew nutrient medium of the following composition, g/l:

Na₂HPO₄•12H₂O 6.20 KH₂PO₄ 1.65 glucose 20.0-60.0 carbamide 10.0-24.0MgSO₄•7H₂O 0.8-1.0 ZnSO₄•7H₂O 0.08-0.4  a calcium salt 0.2-0.6 a cobaltsalt  0.04-0.085 FeSO₄•7H₂O in a chelate complex form 0.025-0.05 distilled water to 1 l pH 7.0-7.4.

The nutrient medium in the second vessel may comprise one of followingsalts as a calcium salt: CaHPO₄.2H₂O, Ca(H₂PO₄)₂.2H₂O, Ca₃ (PO₄)₂,CaCl₂.2H₂O, CaCO₃, CaSO₄, Ca(C₃H₅O₃)₂.3H₂O, Ca (HOCH₂ (CHOH)₄COO)₂.H₂O;and one of following salts as a cobalt salt: CoCl₂.6H₂O, CoSO₄.7H₂O.

Glucose, carbamide and cobalt salts can be introduced in the nutrientmedium in one portion or in several portions during the cell growth.

The value of optical density in the second vessel may be brought to0.1-0.3units. The process may be continued for 48-120 hours at atemperature of 26-31° C., an aeration of 0.5-1.0 volume of air/volume ofmedium per a minute until the achievement of an optical density of thesuspension in the range of 36-40 and a pH of 7.5-7.8. After the end ofthe process, thebiomass is separated by any known method such ascentrifugation, floculation, flotation, sedimentation with the followingfiltration. Implementation of this method of cultivating strainRhodococcus aetherivorans VKM Ac-2610D can give a cell yield of 10-18g/1 with the activity of nitrile hydratase enzyme of 250-332 U/mg. Thebiomass can be used later in processes of producing acrylamide.

Testing the activity of nitrile hydratase enzyme may be carried out asfollows: a suspension of cells is prepared in 0.01 M phosphate buffer,pH 7.6, at a concentration of 0.04-0.06 mg of cells (on a dry weightbasis)/ml. The acrylonitrile substrate in the amount of 25 μl isintroduced into 1 ml of suspension. A transformation reaction is carriedout in a water bath at a temperature of 20-25° C. and with a stirringfor 10 min. The reaction is stopped by adding 50 μl of 6H HCl. Bacterialcells are separated by centrifugation. The concentration of acrylamidein a supernatant is determined by a spectrophotometric or gaschromatographic method. Activity measurements of nitrile hydratasedescribed herein and in the Examples have been performed at atemperature of 20° C. unless otherwise indicated.

A unit of a specific nitrile hydratase activity (U/mg) is an amount ofthe enzyme catalyzing generation of 1 μM of acrylamide per unit of time(1 minute) in 1 mg of cells (on a dry weight basis). A unit of a totalnitrile hydratase activity (U/ml) is an amount of enzyme units containedin 1 ml of a culture broth.

In detail, the method of producing acrylamide may be carried out asfollows.

Cells of bacterial strain Rhodococcus aetherivorans VKM Ac-2610Dobtained as indicated in the description of the method of cultivation,may be separated from the culture broth according to any known method,washed with desalted water, pH 7.0-7.6. The amount of water which can beused for washing may be 1-10 volumes/volume of cells. The washed cellscan be suspended in a tap or distilled water, pH 6.8-8.4, at about400-500 g of cells (on dry weight basis) per 1 ton of the final product,i.e. 45-49% solution of acrylamide may be prepared depending on theintended concentration of a final product and time of synthesis. Thewater suspension of cells can be placed in a thermostatic reactor. Thereaction of acrylamide synthesis may be carried out in the range of pH6.8-8.4. Acrylonitrile may be loaded into the reaction solution duringtransformation so that its concentration in the solution is no more than0.5%. The reaction mixture may be constantly stirred. The temperature ofthe reaction solution can be maintained in the range of 10-23° C. Timeof reaction may be 5-8 hours. Acrylamide and acrylonitrile in thereaction solution may be analyzed by any known method, for exampleliquid or gas chromatography, spectrophotometry, refractometry. Theanalysis can be carried out at least once an hour. The process can bestopped after a sudden drop of a rate of acrylonitrile hydrolysis. Aftertermination of the process cells of the biocatalyst may be separatedfrom the reaction mixture by any known method, for example by afloculation, filtration, flotation, centrifugation. The process can givesolutions having a concentration of acrylamide of e.g. 45-49%.

A skilled person will understand that the methods may be scaled so as tocultivate large bio-masses of the bacterial strain and to produce largeamounts of acrylamide in an industrial scale e.g. by utilizingindustrial fermentors and reactors having a large volume.

EXAMPLES

In the following, the present invention will be described in moredetail. The description below discloses some embodiments and examples ofthe invention in such detail that a person skilled in the art is able toutilize the invention based on the disclosure. Not all steps of theembodiments are discussed in detail, as many of the steps will beobvious for the person skilled in the art based on this specification.The following examples were carried out in a small-scale testinglaboratory; however, a person skilled in the art is able to scale theexamples as desired.

Example 1 Isolation of a bacterial strain Rhodococcus aetherivorans VKMAc-2610D being a producer of a nitrile hydratase enzyme

The strain is isolated from a wastewater of acrylamide andpolyacrylamide manufacture.

Composition of wastewater:

Acrylamide  6.0 g/l; Acrylonitrile  3.3 g/l; Cu²⁺ 1.25 mg/l Zn²⁺ 1.93mg/l Fe³⁺ 0.43 mg/l Sorbital C-20 traces.

For isolation of the strain a selective medium was used representing anagarized wastewater having the above composition supplemented with 0.9g/l of Na₂HPO₄.12H₂O, 0.5 g/l of KH₂PO₄, 1.0 g/l of MgSO₄.7H₂O, 0.5 g/lof an yeast extract. Plates with the selective medium were seeded with alawn of 0.1 ml of a wastewater and cultivated for 72-96 hours. The growncolonies were reseeded twice on a meat and peptone agar to test purityand then they were analyzed for the ability to transform acrylonitrileto acrylamide. For this purpose the tested strains were grown inbacteriological tubes having a volume of 40 ml, ⅛-full with a mediumhaving the following composition (g/l):

Na₂HPO₄•12H₂O 0.9 KH₂PO₄ 0.5 Glucose 20.0 Carbamide 12.0 CoCl₂•6H₂O 0.02MgSO₄•7H₂O 1.0 Yeast extract 1.0 pH 7.0-7.4

Cultivation was carried out for 2 days with stirring, at a temperature28-30° C. The obtained cell suspension was centrifuged for 1 minute at15000 rpm. Cells were washed with 0.01 M phosphate buffer, pH 7.6,resuspended in 1 ml of the same buffer and then nitrile hydrataseactivity was measured as described in the test. This results in thestrain having a nitrile hydratase activity of 95 U/mg.

Example 2 A Method of Cultivation of the Bacterial Strain Rhodococcusaetherivorans VKM Ac-2610D in Erlenmeyer Flasks in Laboratory Conditions

Cells of the strain were grown on a meat and peptone agar slope for 36hours at 28° C. The obtained biomass was washed out with a sterilephysiological solution, pH 7.0-7.4, and seeded for a precultivation intoa liquid nutrient medium of the following composition, (g/l):

Na₂HPO₄•12H₂O 6.06 KH₂PO₄ 1.3 Glucose 12.0 Carbamide 4.0 MgSO₄•7H₂O 1.0ZnSO₄•7H₂O 0.1 CaCl₂•2H₂O 0.2 CoCl₂•6H₂O 0.02 FeSO₄•7H₂O 0.025 Ascorbicacid 0.05 Distilled water to 1 liter. pH 7.0-7.4

The precultivation was carried out in Erlenmeyer flasks at 28-30° C.,with a circular stirring of 120-160 rpm for 24-48 hours. This results ina cell suspension having an optical density of 4.6 units at λ 540 nm,1=5 mm. The obtained suspension in the amount of 2 ml was asepticallyseeded into a medium for cultivation having the following composition,g/l:

Na₂HPO₄•12H₂O 6.20 KH₂PO₄ 1.65 Glucose 40.0 Carbamide 24.0 MgSO₄•7H₂O1.0 ZnSO₄•7H₂O 0.3 CaHPO₄•2H₂O 0.24 CoCl₂•6H₂O 0.06 FeSO₄•7H₂O 0.05Ascorbic acid 0.10 Distilled water to 1 liter. pH 7.0-7.4

The cultivation was carried out in Erlenmeyer flasks having a volume of300 ml, ⅙-full with the medium, for 4 days with a circular stirring (160rpm), at a temperature of 28-30° C. After completion of the cultivationa concentration of cells and a nitrile hydratase activity in samples wasdetermined. The yield of cells after 96 hours of cultivation was 16.0g/l, a specific nitrile hydratase activity was 332 U/mg at 20° C. and521 U/mg at 25° C., a total nitrile hydratase activity was 5312 U/ml at20° C. and 8336 U/ml at 25° C.

Determination of activity at 25° C. was carried out for comparison witha strain Rhodococcus ruber GT.

To confirm the scalability of the method of cultivation and apossibility of the industrial use of the bacterial strain Rhodococcusaetherivorans VKM Ac-2610D, a cultivation of this strain in a fermenterhaving a volume of 3 liters was carried out.

Example 3 Cultivation of a Bacterial Strain Rhodococcus aetherivoransVKM Ac-2610D in a Fermenter

An inoculum representing a suspension of cells in a culture medium wasprepared during 28 hours. For this purpose cells were grown with acircular stirring (160 rpm), at a temperature of 28-30° C. in Erlenmeyerflasks having a volume of 300 ml ⅙-full with the medium having thefollowing composition, (g/l):

Na₂HPO₄•12H₂O 6.06 KH₂PO₄ 1.3 Glucose 12.0 Carbamide 4.0 MgSO₄•7H₂O 1.0ZnSO₄•7H₂O 0.1 CaCl₂•2H₂O 0.2 CoCl₂•6H₂O 0.02 FeSO₄•7H₂O 0.025 EDTA 0.05Distilled water to 1 liter. pH 7.0-7.4

This resulted in a cell suspension having an optical density of 6 unitsat λ 540 nm, 1=5 mm. 100 ml of the obtained cell suspension were usedfor inoculation of a laboratory fermenter having a volume of 3 liters(filled with 1.5 l of the medium).

A composition of the medium for the fermenter, (g/l):

Na₂HPO₄•12H₂O 6.20 KH₂PO₄ 1.65 Glucose 40.0 Carbamide 22.0 MgSO₄•7H₂O1.0 ZnSO₄•7H₂O 0.25 CaHPO₄•2H₂O 0.24 CoCl₂•6H₂O 0.05 FeSO₄•7H₂O 0.05EDTA 0.10 Distilled water to 1 liter. pH 7.0-7.4

The cultivation was carried out at a temperature of 28° C., an aerationof 0.5-1.0 volumes of air/volume of medium per a minute, with acontinuous stirring of 560 rpm. Periodically, once at six hours, samplesof the culture broth from a fermenter were taken to determine pH, ayield and nitrile hydratase activity of cells. The obtained data arepresented in the table.

Fermentation period, hours 0 6 12 18 24 30 36 pH 7.11 6.95 6.91 6.886.87 6.88 6.87 Yield of 0.1 0.4 1.3 2.4 3.7 5.5 7.1 cells, g/l (on dryweight basis) Specific nitrile — 40 34 55 126 185 219 hydrataseactivity, U/mg Fermentation period, hours 42 48 54 60 66 70 pH 6.86 6.856.92 6.97 7.06 7.51 Yield of 8.2 9.9 12.6 13.9 16.4 18.1 cells, g/l (ondry weight basis) Specific nitrile 236 249 256 267 274 280 hydrataseactivity, U/mg

After 70 hours of cultivation the yield of cells was 18.1 g/l, thespecific nitrile hydratase activity of cells was 280 U/mg (at 20° C.) or440 U/mg at 25° C., the total nitrile hydratase activity was 5058 U/ml(at 20° C.) or 7964 U/ml at 25° C.

Example 4 Preparation of a Concentrated Acrylamide Solution in aLaboratory Reactor Using the Cells Produced by Cultivation in Flasks

66.2 g of a cell suspension of the bacterial strain Rhodococcusaetherivorans VKM Ac-2610D in tap water having a nitrile hydrataseactivity of 270 U/mg were placed into a 150 ml thermostatic reactorsupplied with a magnetic mixer and thermometer; thus, the reactorcontained 30 mg (on dry weight) of the biocatalytic cells. The cellswere produced as described in Example 2. pH of the reaction solution was7.6. 24.2 g of acrylonitrile were loaded in the reactor at continuousstirring and a temperature of 17-22° C. over the whole period ofsynthesis so that an acrylonitrile concentration in the solution was nomore than 0.3%. Acrylamide and acrylonitrile in the solution wasanalyzed by a gas chromatography method with a periodicity of once anhour. After 7.0 hours of synthesis the reaction was stopped because arate of acrylonitrile hydrolysis decreased. Cells were separated fromthe reaction solution by centrifugation. The concentration of acrylamidein the solution was 49%, residual acrylonitrile was absent.

To confirm the possibility of using the strain Rhodococcus aetherivoransVKM Ac-2610D in a process of the industrial acrylamide preparation andthe scalability of the claimed technology, a synthesis of acrylamidewere carried out in an apparatus having a volume of 3 liters, which isan analogue of the industrial reactor.

Example 5 Synthesis of a Concentrated Acrylamide Solution in a 3 lReactor Using the Cells Obtained by The Cultivation in Flasks

1.4 l of a cell suspension of the strain Rhodococcus aetherivorans VKMAc-2610D in the desalted water having the activity of a nitrilehydratase enzyme of 254 U/mg were loaded into a 3 liter reactor suppliedwith a mechanical mixer and jacket for a heat removal, andthermostatically controlled in a range of temperatures 20-22° C.; thus,the reactor comprised 1 g (on dry weight) of the biocatalytic cells. Thecells were obtained as described in example 2. pH of the reactionsolution was 7.4. Acrylonitrile was introduced into the reactionsolution as it transformation occurs so that an acrylonitrileconcentration, both initial and current, was no more than 0.5%. After 5hours of the reaction, an acrylamide solution having a concentration of47% was obtained. Then the reaction was stopped because of the sharpdecrease of the acrylonitrile hydrolysis rate.

Example 6 Preparation of a concentrated acrylamide solution in thereactor having a volume of 3 liters using the cells produced bycultivation in a fermenter

1.4 liters of a cell suspension of the strain Rhodococcus aetherivoransVKM Ac-2610D in the desalted water having the activity of nitrilehydratase enzyme of 280 U/mg were loaded into a 3 liter reactor suppliedwith a mechanical mixer and a jacket for a heat removal andthermostatically controlled in the range of temperatures of 14-23° C.;thus, the reactor comprised 1 g (on dry weight) of the biocatalyticcells. The cells were obtained as described in Example 3. pH of thereaction solution was 7.8. Acrylonitrile was introduced into thereaction solution as its transformation occurs so that an acrylonitrileconcentration, both initial and current, was no more than 0.1%.Acrylamide and acrylonitrile in the solution were analyzed by a gaschromatography method with a periodicity of once in hour. After 7.0hours the reaction was stopped because of a decrease of theacrylonitrile hydrolysis rate. The concentration of acrylamide in thesolution (i.e. the final product) was 49%. A residual acrylonitrile insolution was absent.

Example 7 Preparation of a concentrated acrylamide solution in thereactor having a volume of 1 liters using the cells produced bycultivation in a fermenter having a volume of 24 liters

A biomass of the bacterial strain was grown as described in the previousexamples, except a fer-menter having a volume of 24 liters was used. Thebiomass was used to prepare acrylamide as described in the previousexamples using a reactor having a volume of 1 liter. The concentrationof acrylamide in the final product obtained was 49.5%. a concentrationof 47-49%

It is obvious to a person skilled in the art that with the advancementof technology, the basic idea of the invention may be implemented invarious ways. The invention and its embodiments are thus not limited tothe examples described above, instead they may vary within the scope ofthe claims.

The invention claimed is:
 1. A method for producing acrylamide, themethod comprising hydration of acrylonitrile using a biomass of abacterial strain belonging to the genus Rhodococcus and having a nitrilehydratase activity, wherein hydration is carried out at a workingconcentration of acrylonitrile which is no more than 0.5%, and thebacterial strain is Rhodococcus aetherivorans VKM Ac-2610D.
 2. Themethod of claim 1, wherein acrylonitrile is mixed in an aqueoussuspension of the biomass of the bacterial strain to form a reactionsolution; and hydration is carried out so that the working concentrationof acrylonitrile in the reaction solution is maintained at no more than0.5%.
 3. The method of claim 1, wherein hydration is followed byisolation of acrylamide.
 4. The method of claim 1, wherein the biomassof the bacterial strain is at about 100-1000 g, or 200-1000 g, or200-800 g, or 300-600 g, or about 400-500 g, or less than 1000 g, orless than 500 g, or less than 400 g on a dry weight of strain per 1 tonof a final product.
 5. The method of claim 4, wherein the final productcontains acrylamide at a concentration of at least 40%; or at least 41%;or at least 42%; or at least 43%; or at least 44%; or 40-55%; or 41-55%;42-55%; or 43-55%; or 44-55%; or at least 45%; or at least 46%; or atleast 47%; or 45-55%; or 45-54%; or 45-53%; or 45-52%; or 45-51%; or45-50%; or 45-49%; or 46-55%; or 46-54%; or 46-53%; or 46-52%; or46-51%; or 46-50%; or 46-49%; or 47-55%; or 47-54%; or 47-53%; or47-52%; or 47-51%; or 47-50%; or 47-49%.
 6. The method of claim 1,wherein hydration of acrylonitrile is carried out at a temperature of10-23° C.
 7. The method of claim 1, wherein hydration of acrylonitrileis carried out at a pH of 6.8-8.4.
 8. The method of claim 1, wherein thebiomass of the bacterial strain is at about 100-1000 g on a dry weightof strain per 1 ton of a final product.
 9. The method of claim 1,wherein the biomass of the bacterial strain is at about 200-1000 g on adry weight of strain per 1 ton of a final product.
 10. The method ofclaim 1, wherein the biomass of the bacterial strain is at about 200-800g on a dry weight of strain per 1 ton of a final product.
 11. The methodof claim 1, wherein the biomass of the bacterial strain is at about300-600 g on a dry weight of strain per 1 ton of a final product. 12.The method of claim 1, wherein the biomass of the bacterial strain is atabout 400-500 g on a dry weight of strain per 1 ton of a final product.13. The method of claim 1, wherein the biomass of the bacterial strainis less than 1000 g on a dry weight of strain per 1 ton of a finalproduct.
 14. The method of claim 1, wherein the biomass of the bacterialstrain is less than 500 g on a dry weight of strain per 1 ton of a finalproduct.
 15. The method of claim 1, wherein the biomass of the bacterialstrain is less than 400 g on a dry weight of strain per 1 ton of a finalproduct.
 16. The method of claim 4, wherein the final product containsacrylamide at a concentration of 47-55%.
 17. The method of claim 4,wherein the final product contains acrylamide at a concentration of47-54%.
 18. The method of claim 4, wherein the final product containsacrylamide at a concentration of 47-52%.
 19. The method of claim 4,wherein the final product contains acrylamide at a concentration of47-52%.
 20. The method of claim 4, wherein the final product containsacrylamide at a concentration of 47-49%.